The ability to study biological processes at the level of a single molecule is of great scientific importance. One of the primary goals of molecular and cellular biology research is to understand the biophysical properties of biological systems, e.g. nucleic acids and proteins, and their dynamic interactions with their environment. Traditional visualisation of single molecules using techniques such as X-ray crystallography and NMR spectroscopy are limited by their static observations and require ensemble imaging that is not strictly representative of a single molecule. New techniques are therefore required that allow single molecules to be monitored with high sensitivity and resolution.
Recent advances in imaging techniques have made it possible to observe the dynamic behaviour of a single molecule (see, for example, Ha et al., Proc Natl Acad Sci USA. 1999;96(3):893-8). However, these approaches have generally been based on indirect methods relying on the detection of fluorescent dyes. These dye systems suffer from a number of drawbacks, such as blinking, spiking and photobleaching, which limit the observations which can be made.
Photobleaching is a well documented phenomenon in fluorescent dye systems. All dye systems have the ability to absorb a limited number of photons before the fluorescent dye is no longer visible to the observer, i.e. it has been photobleached. If the dye is conjugated to a molecule under study, the molecular kinetics, dynamics and reaction pathway under observation will no longer be visible. This is a particular problem in the study of polynucleotide processive enzymes, where conformational changes resulting from the interaction with a polynucleotide can be monitored to allow polynucleotide sequencing, as outlined in WO-A-99/05315.
Photobleaching is a problem in all high-resolution assays involving fluorescent dye molecules (J. Histochem. Cytochem., 1999; 47:1179), especially when such dyes require repeated excitation at high frequency in order to obtain the information about the fluorophore required, for example Fluorescent Lifetime Imaging Spectroscopy (FLIMS) and Flourescent Polarisation Anisotropy measurements.
Blinking is a phenomena that has been observed in conjunction with photobleaching and is a considerable limitation in the context of experiments requiring fluorescent labels. Blinking events, a particular problem in relation to quantum dots or nanocrystal dyes, are unpredictable and can add error to experimental data. Such ‘quantum’ events are of particular relevance to single molecule measurements.
Furthermore, problems are associated with attaching “large” fluorescent labels to pharmaceutically important molecules. These problems are enhanced in small molecule systems where the labels are larger with respect to the molecule under study.
Non-fluorescence based techniques such as Surface Plasmon Resonance (SPR) and Evanescent Spectroscopy are therefore preferred for detecting single molecules. However, these techniques have, to date, typically operated on a multi-molecule scale due to a lack of sensitivity. Attempts to improve sensitivity have been made by monitoring changes in molecular dielectric information directly, to produce dynamic conformational information. One such approach is detailed in WO-A-01/18246, wherein a device is used to measure changes in the capacitance of the fluid, gas, molecule, particle or cell under study as it passes through the device. However, the transient nature of this system allows only relative flow-through data to be obtained, and not conformational data.
The ability to monitor real-time conformational changes of a single molecule is particularly applicable in the field of polynucleotide sequencing, where it is desirable to monitor the polynucleotide processive enzyme to detect the interaction(s) with the target polynucleotide, for example the addition of each successive base. This removes the need for labelled nucleotide substrates (particularly fluorescently-labelled), which can interfere with the catalytic activity of the enzyme, reducing the sequencing rate and increasing the mis-incorporation rate. A system capable of real-time sequencing without the use of labelled nucleotides or nucleotide analogues would therefore offer the considerable advantages of faster sequencing, longer read length and lower error rates.
New techniques are therefore required that are able to monitor and record single molecule dynamic conformational changes with high resolution and sensitivity, and avoid the problems associated with fluorescence. Such a technique could be used for protein characterisation, ultra-sensitive chemical analysis and rapid DNA sequencing.